摘要
目的利用原核表达系统表达华南地区分离的三源重组H9N2亚型禽流感病毒(Avian influenza virus,AIV)的神经氨酸酶(NA)蛋白,并制备兔多克隆抗体。方法将1株H9N2亚型AIV的NA基因克隆到pET-32a载体,构建NA原核表达质粒,并转化至E.coli Rosetta(DE3)感受态细胞进行蛋白表达。优化IPTG浓度、诱导时间和温度,提高蛋白表达量。利用镍柱纯化重组NA蛋白,并免疫新西兰兔,制备兔抗NA蛋白多克隆抗体通过Western blot和间接免疫荧光试验(Indirect immunofluorescence assay,IFA)验证多克隆抗体的特异性。通过间接酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)测定多克隆抗体的效价。结果成功构建了pET-32a-H9N2-NA原核表达质粒;经SDS-PAGE和Western blot验证,表达的重组NA蛋白大小约70 ku,符合预期;对诱导条件进行优化,确定37℃,终浓度为1.2 mmol/L的IPTG,诱导时长6 h为最佳条件;制备的兔抗NA蛋白多克隆抗体的Western blot和IFA的结果显示,该多克隆抗体能和H9N2亚型AIV结合,特异性好;间接ELISA测得兔抗NA蛋白多克隆抗体的效价达到了1∶409600。结论本研究表达的NA蛋白,制备的兔抗NA蛋白多克隆抗体具有特异性,效价高,为NA蛋白的ELISA检测方法的建立和疫苗研制提供帮助。
Objective This study aimed to express the neuraminidase(NA)protein of a triple-reassortant H9N2 subtype avian influenza virus(AIV)isolated from South China using a prokaryotic expression system and to generate rabbit polyclonal antibodies.Methods The NA gene from an H9N2 AIV strain was cloned into the pET-32a vector to create an NA prokaryotic expression plasmid,which was then introduced into E.coli Rosetta(DE3)competent cells for protein expression.Optimization of IPTG concentration,induction time,and temperature was conducted to enhance protein expression.The recombinant NA protein was purified using a nickel column,and New Zealand rabbits were immunized to produce rabbit anti-NA protein polyclonal antibodies.The specificity of the polyclonal antibodies was confirmed through Western blot and Indirect Immunofluorescence Assay(IFA),while the titer of the polyclonal antibodies was evaluated using Enzyme-Linked Immunosorbent Assay(ELISA).Results The construction of the pET-32a-H9N2-NA prokaryotic expression plasmid was successful.Analysis via SDS-PAGE and Western blot validated the expression of the recombinant NA protein,which exhibited the expected size of approximately 70 ku.Optimal induction conditions were determined to be 37℃,a final IPTG concentration of 1.2 mmol/L,and an induction time of 6 hours.Western blot and IFA results of the generated rabbit anti-NA protein polyclonal antibodies demonstrated strong specificity in binding to the H9N2 AIV.The titer of the rabbit anti-NA protein polyclonal antibodies reached 1∶409600 as determined by indirect ELISA.Conclusion The expressed NA protein and the resulting rabbit anti-NA protein polyclonal antibodies exhibited high specificity and titer,thus facilitating the establishment of an ELISA detection method for NA protein and contributing to vaccine development efforts.
作者
梁志鹏
刘静
刘闰栀
张新宇
何杰珩
陈高婕
闫战飞
池仕红
袁生
郭锦玥
梁昭平
黄淑坚
温峰
LIANG Zhipeng;LIU Jing;LIU Yunzhi;ZHANG Xinyu;HE Jieheng;CHEN Gaojie;YAN Zhanfei;CHI Shihong;YUAN Sheng;GUO Jinyue;LIANG Zhaoping;HUANG Shujian;WEN Feng(College of Life Science and Engineering,Foshan University,Foshan 528225,Guangdong,China;College of Veterinary Medicine,South China Agricultural University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2024年第6期625-630,共6页
Journal of Pathogen Biology
基金
广东省基础与应用基础研究基金(No.2022A1515012462,2024A1515010836)
广东省教育厅科研项目(No.2022KTSCX124)
广东大学生科技创新培育专项资金资助项目(No.pdjh2023b0547)
佛山科学技术学院学生学术基金项目(No.xsjj202309kja01,xsjj202309zrb06)。
关键词
禽流感病毒
H9N2亚型
神经氨酸酶
原核表达
多克隆抗体
avian influenza virus
H9N2 subtype
neuranimidase
prokaryotic expression
polyclonal antibody
